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Filamentous structures containing a keratin-like protein in spermatozoa of an insect, Bacillus rossius
JOURNAL OF ULTRASTRUCTURE RESEARCH
86, 86-92 (1984)
Filamentous Structures Containing a Keratin-like Protein in Spermatozoa of an Insect, Bacillus rossius B. BACCETTI,* A. G. BURRINI,* G. GABBIANI,t P. LEONCINI,~ AND E. RUNGGER-BRANDLEt *Institute of Zoology, University of Siena, Italy, tDepartment of Pathology, University of Geneva, Switzerland, and "~Center of Research, ISVT "Sclavo, '" Siena, Italy Received November 15, 1983 Spermatozoa of a phasmid insect, Bacillus rossius, contain, in the whole length of the tail, two longitudinal crystalline bodies flanking the axoneme and made up of a texture of 10-nm filaments. These filaments are resistant to nonionic detergent extraction (Triton X- 100) and appear particularly evident after deep-etching. SDS-polyacrylamide gel electrophoresis, immunoblotting, and immunochemical methods, at both the light and the electron microscopic level, show that they share antigenic determinants with the vertebrate cytokeratins. All these properties suggest that these filaments are members of the cytokeratin family. The occurrence of keratin in invertebrates is discussed.
The cytoskeleton of most animal cells contains, in addition to microfilaments and microtubules, a third type of fibrous structure with a diameter of 7-11 nm, the intermediate-sized filaments. Although morphologically similar, these filaments display pronounced tissue specificity of biochemical composition (Lazarides, 1980, 1982) which makes them useful markers in studying cell differentiation phenomena (Lazarides, 1982; Osborn and Weber, 1982). Among vertebrates, homologies for some of the constituent proteins of intermediatesized filaments, in particular vimentin and desmin, were found to exist in mammals, birds, reptiles, amphibia, and fishes. First, Franke et al. (1979a, b) found an immunologic relatedness, after that Geisler and Weber (1981) described biochemical similarities, and finally Nelson and Traub (1982) showed some similarities in sizes and in binding to polyanions. These observations indicate evolutionary conservation of at least some domains of these molecules. Also, the heterogeneous family of cytokeratins (Franke et al., 1981) from vertebrate epithelial cells show a conservative feature in molecule. Franke et aL (1978) found an im0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
munological relatedness across species from frog to man; a view substantiated by peptide mapping according to Schiller et al. (1982). Intermediate-sized filaments have been observed by electron microscopy in invertebrate groups as distinct as protozoa (Schrevel and Philippe, 1981), mollusks (Kessel and Beams, 1981) and annelids (Gilbert and Newby, 1975). However, their isolation and characterization has been, until now, restricted for practical reasons to the neurofilaments ofmoUusks (Lasek et al., 1979) and annelids (Lasek et al., 1979; Eng et al., 1980). For squid brain filaments, it was shown that the basic assembly process, as it may be induced under in vitro conditions, is similar to what has been described for mammalian filaments, although there exist differences in solubility and polypeptide compositions (Lasek et al., 1979). Moreover, evidence has been presented that cultured Kc cells of Drosophila melanogaster contain cytoplasmic proteins that have at least one immunologic determinant in common with the intermediate-sized proteins of vertebrate vimentin and desmin (Falkner et al., 1981). Moreover, Fuchs et al. (1981) at first were not able to find Dro86
KERATIN-LIKE FILAMENTS IN INSECT SPERMATOZOA s o p h i l a D N A h y b r i d i z i n g w i t h c D N A seq u e n c e s specific for k e r a t i n s , b u t m o r e rec e n t l y s u c c e e d e d i n this d e m o n s t r a t i o n . I n s p e r m a t o z o a o f i n v e r t e b r a t e s , the occ u r r e n c e o f f i b r o u s s t r u c t u r e s w i t h the diameter of intermediate-sized filaments has b e e n d e s c r i b e d (Kessel a n d B e a m s , 1981), but i m m u n o l o g i c a l a n d biochemical chara c t e r i z a t i o n s h a v e n o t yet b e e n p e r f o r m e d . F o r o u r studies, we h a v e c h o s e n the sperm a t o z o a o f the insect B a c i l l u s rossius, w h i c h we h a v e p r e v i o u s l y s h o w n to c o n t a i n u l t r a s t r u c t u r a l l y well d e f i n e d f i b r o u s s t r u c t u r e s f o r m i n g two l o n g i t u d i n a l c r y s t a l l i n e b o d i e s a l o n g t h e tail (Baccetti et aL, 1973) region. T h e s e structures, called " a c c e s s o r y b o d i e s " (Baccetti a n d Afzelius, 1976) are c h a r a c t e r istic o f the s p e r m i n s o m e insects. A n a n a l ysis o f these " a c c e s s o r y b o d i e s " b y m e a n s o f b i o c h e m i c a l a n d i m m u n o c h e m i c a l techn i q u e s suggest t h a t t h e y are c o m p o s e d at least i n p a r t o f k e r a t i n - l i k e m o l e c u l e s .
MATERIALS AND METHODS Seminal vesicles were excised from adult males of B. rossius (Insecta, Phasmatodea). Alive spermatozoa were obtained by squeezing vesicles in Hoyle's solution (0.375 g KC1; 3.75 g NaC1; 0.110 g CaC12;0.205 g MgCI; 0.170 g NaHCO3; and 0.415 g NaH2PO4 added to 500 ml distilled H20). Antibodies
Antibodies against bovine muzzle prekeratin (Franke etaL, 1978; Franke etal., 1980; Gabbiani etaL, 1981), chicken gizzard, human uterine desmin (Gabbiani et aL, 1981, 1982; Denk et al., 1983), or cultured human fibroblast vimentin (Denk et aL, 1983; Gabbiani et aL, 1982; Franke et al., 1979) were obtained in guinea pigs as previously described (Denk et aL, 1983; Gabbiani et aL, 1982). The specificity of the keratin antibodies against bovine muzzle prekeratin was tested by immunoblotting(Towbin et al., 1979). Moreover, an antibody against bovine hoofprekeratin, affinity-purified according to Nagle et al. (1983), was used. Immunological Reaction in SDS-PAGE
For Bacillus "sperm extract," spermatozoa were washed in PBS, rinsed briefly in 10 mM Tris-HC1, pH 7.4, and dissolved in sample buffer containing 3% sodium dodecyl sulfate, 5% /3-mercaptoethanol, and 1 m_M phenylmethylsulfonylfluoride (PMSF). Gel electrophoresis was essentially according to Laemmli (1970) using gradient gels of 5-20% acrylamide. Proteins were
87
transferred electrophoretically on nitrocellulose paper sheets (Towbin et al., 1979) and incubated with either the antiserum against prekeratin or rabbit (preimmune) serum (dilution 1:40). A goat anti-rabbit Ig fraction coupled with peroxidase (Nordic, Tilburg, Netherlands, 3 ~1/5 ml) was used as second antibody. Blots were stained with dianisidine (Fluka, Buchs, Switzerland). Immunofluorescence Microscopy
All steps were carried out at room temperature. Drops of living spermatozoa were applied to polylysine (0.1%) coated glass slides. Cells were allowed to settle for 15 rain, then fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.1% saponin in PBS (15 min). After a brief rinsing in PBS, the first antiserum was added at a dilution 1:10 (20 min). After washing in PBS, incubation in FITC-labeled rabbit anti-guinea pig or antirabbit 3,-globulins (dilution 1:5 and 1:20, respectively; Behringwerke, Marburg, FRG) was for 15 min. Specimens were observed and photographed using a Zeiss photomicroscope equipped with epifluorescence illumination. Electron Microscopy Routine technique. Seminal vesicles dissected from adult males of B. rossius were fixed in Karnovsky's fixative (Karnovsky, 1965) in 0.1 M cacodylate buffer, pH 7.2, for 2 hr at 4°C, rinsed in the same buffer overnight, postfixed in 1% buffered cacodylate OsO4 for 1 hr at 4°C, stained en bloc with 1% aqueous uranyl acetate, dehydrated in ethanol, and embedded in Epon. Sections were stained with uranyl acetate and lead citrate, and observed with a Philips 300 or 301 electron microscope. Briefly sonicated and unfixed material was negatively stained with 2% phosphotungstic acid. Extractions. Mature spermatozoa obtained from seminal vesicles orB. rossius were extracted for 20 min at room temperature with 1% Triton X-100 in Hoyle's solution (pH 7.2) or with 0.5% Triton X-100 in Hoyle's solution containing 1.5 M KC1 and 5 mM EGTA. Spermatozoa collected by centrifugation were then fixed and processed as described above. lmmunocytochemicalstaining. Mature spermatozoa were fixed and permeabilized with 0.02% saponin in PBS according to the method of Willingham and Yamada (1979). They were then stained by the indirect method, using initially a 1:10 dilution of guinea pig antiprekeratin antiserum in PBS-saponin, then a 1:10 dilution of PBS-saponin of a rabbit anti-guinea pig IgG fraction conjugated with horseradish peroxidase (Nordic Immunological Laboratories, Tilburg). Cells were fixed again with 3% glutaraldehyde in PBS and rinsed in PBS for 30 min. Horseradish peroxidase was detected by diaminobenzidine(Graham and Karnovsky, 1966). After postfixation in 1% OsO4 in PBS for 30 min at 4°C, the cells were dehydrated and
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ET AL.
FIGS. l-6. Immunofluorescent staining of prekeratin in B. rossius spermatozoa. In Figs. 2 and 4 the complete tail region appears uniformly marked by the antiprekeratin antibody, while the head (arrows) is unstained (compare with the phase-contrast micrographs in Figs. 1 and 3). In Fig. 5 a control spermatozoon treated with a normal guinea pig serum is shown at phase contrast; no fluorescence is visible (Fig. 6). x 400.
embedded as described above. Alternatively, spermatozoa processed as described for immunofluorescence microscopy, were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 12 h at 4°C and processed for electron microscopy, according to Sharp et al. (1982).
RESULTS
Indirect immunofluorescence microscopy using antibodies directed against mammalian prekeratin, and particularly affinity purified antibodies, revealed bright staining
89
KERATIN-LIKE FILAMENTS IN INSECT SPERMATOZOA
of the whole-tail region of the spermatozoon (Figs. 1-4). This staining was assumed to be specific, since it was not observed in control preparations incubated with preimmane guinea pig serum instead of prekeratin antibody containing serum (Figs. 5, 6). Using antibodies against other components of intermediate-sized filaments, vimentin and desmin, no distinct staining could be localized (not shown). In agreement with the morphological staining pattern, we found after immunoblotting a cross-reaction of the same prekeratin antibodies with two polypepetides present in a total lysate of sperm cells of B. rossius (Fig. 7). They are minor components, since they do not stain with Coomassie blue. The molecular weights of these prekeratin-like polypeptides are a b o u t 60 000 and 68 000 Da and thus within the range o f known vertebrate prekeratins (Franke et aL, 1981; Moll et aL, 1982). Electron microscopic examination revealed two longitudinal elongate organelles in the whole length of the tail region (Fig. 8). They are made up by four juxtaposed laminae, longitudinally oriented with respect to the flagellar major axis. These four laminae are separated from one another by a space 10 nm wide, and consist of longitudinally oriented filaments lying at 10-rim intervals to one another. The 10-nm filaments are particularly evident after sonication and negative staining (Fig. 10). This array of filaments remains well preserved even after treatment with nonionic detergent Triton X-100 and high ionic strength solutions (Fig. 9). When saponin-treated spermatozoa were incubated with antiprekeratin antibodies and subsequently revealed by indirect staining with horseradish peroxidase, an intense staining over the crystalline bodies became visible (Fig. 11). Control incubations remained unstained (Fig. 12). Spermatozoa treated with prekeratin and FITC-conjugated antibodies revealed the same staining pattern at the level of the two crystalline
a
b
c
d
e
m
FIG. 7. Immunologic reaction of total extract of Bacillus sperm resolved by polyacrylamide gel electrophoresis (bands b and d), blotted on nitrocellulose sheets and incubated either with prekeratin antibodies against bovine prekeratin (c) or with normal rabbit serum as a control (e). Note (*) specific reaction of two minor bands from total sperm extract with prekeratin antibodies (c). (a) Markers: V, vimentin; A, Actin.
organelles (Fig. 13) not visible in the controls (Fig. 14). We therefore conclude that the proteins which are detected by crossreacting prekeratin antibodies, are localized in the crystalline bodies of the insect sperm tail. DISCUSSION
We have presented evidence that sperm cells of the insect B. rossius contain two polypeptides which specifically react with keratin antibodies, thus appear to share common antigenic determinants with vertebrate cytokeratins. Moreover, they have the molecular weight within the range of mammalian prekeratins (Franke et al., 1981; Moll et al,, 1982), and form 10-nm filaments, which are insoluble in nonionic detergent and at high ionic strength. In order to localize these polypeptides in the sper-
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W
r p
FIG. 8. Spermatozoon of B. rossius. Cross section of the tail region showing the two enormous crystalline accessory bodies (b) flanking the axoneme (a). FIG. 9. The same region in a Triton-extracted sperm. Accessorybodies (b) appear unchanged, x 100 000. Fit. 10. Negativestaining with PTA of an accessory body ofB. rossius sperm tail. The body appears as a bundle of 10-nm filaments. × 75 000.
m a t o z o o n , we used i m m u n o h i s t o c h e m i c a l methods, at both the light and electron microscopic level. With these methods, we localized these prekeratin-like polypeptides in the crystalline accessory bodies o f the sperm tail, which in fact consist o f a packing o f 10-nm filaments. These bodies are a characteristic o f the s p e r m a t o z o o n o f several insects, they flank the a x o n e m e t h r o u g h o u t almost its whole length, and are particularly
conspicuous in Bacillus, giving to the flagellar m o v e m e n t a pattern o f a double series o f waves (Baccetti et al., 1973). A l t h o u g h i n t e r m e d i a t e - s i z e d filaments have not been as systematically studied in in invertebrates as has been done in higher vertebrates (Osborn and Weber, 1982), they appear to be morphologically similar to, and might share biochemical characteristics with vertebrate 10-nm filaments. For instance,
KERATIN-LIKE FILAMENTS IN INSECT SPERMATOZOA
91
@
@ FIG. 11. Spermatozoon of B. rossius. Cross section of the tail showing accessory bodies (b) strongly stained by the DAB--peroxidase method after antiprekeratin serum incubation, x 100 000. FIG. 12. Control to the above method, after normal guinea pig serum incubation, x 100 000. FIG. 13. Same material, with accessory bodies (b) strongly stained by the FITC-conjugated anti-guinea pig IgG after anti-prekeratin serum incubation, x 100 000. FIG. 14. Control to the above method, after normal guinea pig serum incubation, x 100 000. K c cells of Drosophila melanogaster c o n t a i n h e a t - s e n s i t i v e p r o t e i n s w h i c h a p p e a r to b e constituents of a filamentous cytoplasmic m e s h w o r k ( F a l k n e r et al., 1981). M o n o c l o n a l a n t i b o d i e s r a i s e d a g a i n s t these Dro-
sophila p r o t e i n s c r o s s - r e a c t w i t h v i m e n t i n a n d d e s m i n o f m a m m a l i a n c u l t u r e d cells i n d i c a t i n g the p r e s e n c e o f at least s o m e c o m m o n d e t e r m i n a n t s i n these m o l e c u l e s ( F a l k n e r et aL, 1981). T h e p r e s e n c e o f p r e -
92
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keratin-like polypeptides in spermatozoa o f insects m a y demonstrate that also this group o f proteins, yet displaying p r o n o u n c e d heterogeneity a m o n g m a m m a l s (Franke et al., 1981; Moll et al., 1982), contains some domains firmly conserved during evolution and which are c o m m o n to similar polypeptides in animals as far apart as insects and m a m m a l s . Cytokeratins seem not to be confined in insect sperm: in the same class, tracheal taenidia contain filaments which also can be decorated with keratin antibodies (Baccetti et al., 1984). The failure o f detecting sequences h o m o l o g u e s to c - D N A specific for m a m m a l i a n keratins in Drosophila reported by Fuchs et al. (1981) seemed to be in apparent contrast with our data. But this is due to the fact that the D N A sequence coding for keratins or keratin-like molecules have diverged sufficiently during evolution, so that under conditions o f high hybridization stringency, a m a m m a l i a n probe does not recognize insect sequences. In fact m o r e recently the same authors (Fuchs and Marchuk, 1983) have been able to extend to the g e n o m e o f Drosophila (and o f yeast) sequences related to h u m a n kerRtins cDNAs. It means that the proteins themselves can have remained sufficiently conserved during animal evolution to allow recognition o f at least some antigenic determinants by an antibody raised against bovine keratin.
FALKNER,F. G., SAUMWEBER,H., AND BIESSMANN,H. (1981) J. CellBiol. 91, 175-183. FRANKE, W. W., SCHILLER,D. L., MOLL, R., WINTER, S., SCHMID, E., ENGELBRECHT,I., DENK, H., KREPLER, R., AND PLATZER,B. (1981) J. Mol. Biol. 153,
933-959. FRANKE,W. W., SCHMID,E., WINTER,S., OSBORN,M., ANDWEBER,K. (1979) Exp. Cell Res. 123, 25-46.
FRANKE,W. W., SCHMID,E., FREUDENSTEIN,C., APPELHANS,B., OSBORN,M., WEBER,K., ANDKEENAN,
T. W. (1980) J. Cell Biol. 84, 633--654. FRANKE,W. W., WEBER,K., OSBORN,M., SCHMID,E., AND FREUDENSTEIN,C. (1978) Exp. Cell Res. 116, 429-445. FUCHS, E. V., COPPOCK,S. M., GREEN,H., ANDCLEVELAND,D. W. (1981) Cell27, 75-84. FUCHS, E. V., AND MARCHUK, D. (1983) Proc. Natl. Acad. Sci. USA 80, 5857-5861. GABBIANI, G., KAPANCI, Y., BARAZZONE, P., AND FRANKE,W. W. (1981) Amer. J. Pathol. 104, 206-
216. GABBIANI,G., RUNGGER-BRANDLE,E., DECHASTONAY, C., ANDFRANKE,W. W. (1982) Lab. Invest. 47, 265269. GILBERT,D. S., ANDNEWBY,B. J. (1975) Nature (London) 256, 586-589. GRAHAM,R. C., AND KARNOVSKY,M. J. (1966) J. Histochem. Cytochem. 14, 291-297. KARNOVSKY,M. J. (1965) J. CellBiol. 27, 137A. KESSEL,R. G., ANDBEAMS,H. W. (1981) J. Submicrosc. Cytol. 13, 551-560. LAEMMLI,U. K. (1970) Nature(London) 227, 680-685. LASEK, R.
J.,
KRISHNAN, N.,
AND KAISER-
MAN-ABRAMOF,I. R. (1979) J. Cell BioL 82, 336346. LAZARIDES,E. (1980) Nature (London) 283, 249-256. LAZARIDES,E. (1982) Ann. Rev. Biochem. 51, 219-250. MOLL,R., FRANKE,W. W., SCHILLER,D. L., GEIGER,
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BACCETTI,B., ANDAFZELIUS,B. A. (1976) The Biology
NELSON, W. J., AND TRAUB, P. (1982) J. Cell Sci. 57,
of the Sperm Cell, Karger, Basel. BACCETTI,B., BURRINI,A. G., DALLAI,R., PALLINI,V., PERITI, F., PIANTELLI,F., ROSATI,F., AND SELMI,G. (1973) J. Ultrastruct. Res. (SuppL) 12, 1-73. BACCETTI, B., BURRINI, A. G., GABBIANI, G. AND LEONCZNI,P, (1984) Physiol. Entom., in press. DENK, H., KREPLER, R., ARTLIEB, U., GABBIANI,G., RUNGGER-BRANDLE,E., LEONCINI,P., AND FRANKE, W. W. (1983) Amer. J. Pathol. 110, 193-208. ENG, L. F., LASEK,R. J., BIGBEE,J. W., AND END, D. L. (1980) J. Histochem. Cytochem. 28, 1312-1318.
462. 25-49. OSBORN,M., ANDWEBER,K. (1982) Cell 31,303-306. SHARP, G. A., SHAW,G., AND WEBER, K. (1982) Exp. CellRes. 137, 403-413. SCHREVEL,J., AND PHILIPPE,M. (198 l) Abstr. Eur. Cy-
toskel. Club Conf. Changins-Nyon, 32. TOWBIN, H., STAEHELIN,T., AND GORDON, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. WILLINGHAM, M. C., AND YAMADA, S. S. (1979) J. Histochem. Cytochem. 27, 947-960.
86, 86-92 (1984)
Filamentous Structures Containing a Keratin-like Protein in Spermatozoa of an Insect, Bacillus rossius B. BACCETTI,* A. G. BURRINI,* G. GABBIANI,t P. LEONCINI,~ AND E. RUNGGER-BRANDLEt *Institute of Zoology, University of Siena, Italy, tDepartment of Pathology, University of Geneva, Switzerland, and "~Center of Research, ISVT "Sclavo, '" Siena, Italy Received November 15, 1983 Spermatozoa of a phasmid insect, Bacillus rossius, contain, in the whole length of the tail, two longitudinal crystalline bodies flanking the axoneme and made up of a texture of 10-nm filaments. These filaments are resistant to nonionic detergent extraction (Triton X- 100) and appear particularly evident after deep-etching. SDS-polyacrylamide gel electrophoresis, immunoblotting, and immunochemical methods, at both the light and the electron microscopic level, show that they share antigenic determinants with the vertebrate cytokeratins. All these properties suggest that these filaments are members of the cytokeratin family. The occurrence of keratin in invertebrates is discussed.
The cytoskeleton of most animal cells contains, in addition to microfilaments and microtubules, a third type of fibrous structure with a diameter of 7-11 nm, the intermediate-sized filaments. Although morphologically similar, these filaments display pronounced tissue specificity of biochemical composition (Lazarides, 1980, 1982) which makes them useful markers in studying cell differentiation phenomena (Lazarides, 1982; Osborn and Weber, 1982). Among vertebrates, homologies for some of the constituent proteins of intermediatesized filaments, in particular vimentin and desmin, were found to exist in mammals, birds, reptiles, amphibia, and fishes. First, Franke et al. (1979a, b) found an immunologic relatedness, after that Geisler and Weber (1981) described biochemical similarities, and finally Nelson and Traub (1982) showed some similarities in sizes and in binding to polyanions. These observations indicate evolutionary conservation of at least some domains of these molecules. Also, the heterogeneous family of cytokeratins (Franke et al., 1981) from vertebrate epithelial cells show a conservative feature in molecule. Franke et aL (1978) found an im0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
munological relatedness across species from frog to man; a view substantiated by peptide mapping according to Schiller et al. (1982). Intermediate-sized filaments have been observed by electron microscopy in invertebrate groups as distinct as protozoa (Schrevel and Philippe, 1981), mollusks (Kessel and Beams, 1981) and annelids (Gilbert and Newby, 1975). However, their isolation and characterization has been, until now, restricted for practical reasons to the neurofilaments ofmoUusks (Lasek et al., 1979) and annelids (Lasek et al., 1979; Eng et al., 1980). For squid brain filaments, it was shown that the basic assembly process, as it may be induced under in vitro conditions, is similar to what has been described for mammalian filaments, although there exist differences in solubility and polypeptide compositions (Lasek et al., 1979). Moreover, evidence has been presented that cultured Kc cells of Drosophila melanogaster contain cytoplasmic proteins that have at least one immunologic determinant in common with the intermediate-sized proteins of vertebrate vimentin and desmin (Falkner et al., 1981). Moreover, Fuchs et al. (1981) at first were not able to find Dro86
KERATIN-LIKE FILAMENTS IN INSECT SPERMATOZOA s o p h i l a D N A h y b r i d i z i n g w i t h c D N A seq u e n c e s specific for k e r a t i n s , b u t m o r e rec e n t l y s u c c e e d e d i n this d e m o n s t r a t i o n . I n s p e r m a t o z o a o f i n v e r t e b r a t e s , the occ u r r e n c e o f f i b r o u s s t r u c t u r e s w i t h the diameter of intermediate-sized filaments has b e e n d e s c r i b e d (Kessel a n d B e a m s , 1981), but i m m u n o l o g i c a l a n d biochemical chara c t e r i z a t i o n s h a v e n o t yet b e e n p e r f o r m e d . F o r o u r studies, we h a v e c h o s e n the sperm a t o z o a o f the insect B a c i l l u s rossius, w h i c h we h a v e p r e v i o u s l y s h o w n to c o n t a i n u l t r a s t r u c t u r a l l y well d e f i n e d f i b r o u s s t r u c t u r e s f o r m i n g two l o n g i t u d i n a l c r y s t a l l i n e b o d i e s a l o n g t h e tail (Baccetti et aL, 1973) region. T h e s e structures, called " a c c e s s o r y b o d i e s " (Baccetti a n d Afzelius, 1976) are c h a r a c t e r istic o f the s p e r m i n s o m e insects. A n a n a l ysis o f these " a c c e s s o r y b o d i e s " b y m e a n s o f b i o c h e m i c a l a n d i m m u n o c h e m i c a l techn i q u e s suggest t h a t t h e y are c o m p o s e d at least i n p a r t o f k e r a t i n - l i k e m o l e c u l e s .
MATERIALS AND METHODS Seminal vesicles were excised from adult males of B. rossius (Insecta, Phasmatodea). Alive spermatozoa were obtained by squeezing vesicles in Hoyle's solution (0.375 g KC1; 3.75 g NaC1; 0.110 g CaC12;0.205 g MgCI; 0.170 g NaHCO3; and 0.415 g NaH2PO4 added to 500 ml distilled H20). Antibodies
Antibodies against bovine muzzle prekeratin (Franke etaL, 1978; Franke etal., 1980; Gabbiani etaL, 1981), chicken gizzard, human uterine desmin (Gabbiani et aL, 1981, 1982; Denk et al., 1983), or cultured human fibroblast vimentin (Denk et aL, 1983; Gabbiani et aL, 1982; Franke et al., 1979) were obtained in guinea pigs as previously described (Denk et aL, 1983; Gabbiani et aL, 1982). The specificity of the keratin antibodies against bovine muzzle prekeratin was tested by immunoblotting(Towbin et al., 1979). Moreover, an antibody against bovine hoofprekeratin, affinity-purified according to Nagle et al. (1983), was used. Immunological Reaction in SDS-PAGE
For Bacillus "sperm extract," spermatozoa were washed in PBS, rinsed briefly in 10 mM Tris-HC1, pH 7.4, and dissolved in sample buffer containing 3% sodium dodecyl sulfate, 5% /3-mercaptoethanol, and 1 m_M phenylmethylsulfonylfluoride (PMSF). Gel electrophoresis was essentially according to Laemmli (1970) using gradient gels of 5-20% acrylamide. Proteins were
87
transferred electrophoretically on nitrocellulose paper sheets (Towbin et al., 1979) and incubated with either the antiserum against prekeratin or rabbit (preimmune) serum (dilution 1:40). A goat anti-rabbit Ig fraction coupled with peroxidase (Nordic, Tilburg, Netherlands, 3 ~1/5 ml) was used as second antibody. Blots were stained with dianisidine (Fluka, Buchs, Switzerland). Immunofluorescence Microscopy
All steps were carried out at room temperature. Drops of living spermatozoa were applied to polylysine (0.1%) coated glass slides. Cells were allowed to settle for 15 rain, then fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.1% saponin in PBS (15 min). After a brief rinsing in PBS, the first antiserum was added at a dilution 1:10 (20 min). After washing in PBS, incubation in FITC-labeled rabbit anti-guinea pig or antirabbit 3,-globulins (dilution 1:5 and 1:20, respectively; Behringwerke, Marburg, FRG) was for 15 min. Specimens were observed and photographed using a Zeiss photomicroscope equipped with epifluorescence illumination. Electron Microscopy Routine technique. Seminal vesicles dissected from adult males of B. rossius were fixed in Karnovsky's fixative (Karnovsky, 1965) in 0.1 M cacodylate buffer, pH 7.2, for 2 hr at 4°C, rinsed in the same buffer overnight, postfixed in 1% buffered cacodylate OsO4 for 1 hr at 4°C, stained en bloc with 1% aqueous uranyl acetate, dehydrated in ethanol, and embedded in Epon. Sections were stained with uranyl acetate and lead citrate, and observed with a Philips 300 or 301 electron microscope. Briefly sonicated and unfixed material was negatively stained with 2% phosphotungstic acid. Extractions. Mature spermatozoa obtained from seminal vesicles orB. rossius were extracted for 20 min at room temperature with 1% Triton X-100 in Hoyle's solution (pH 7.2) or with 0.5% Triton X-100 in Hoyle's solution containing 1.5 M KC1 and 5 mM EGTA. Spermatozoa collected by centrifugation were then fixed and processed as described above. lmmunocytochemicalstaining. Mature spermatozoa were fixed and permeabilized with 0.02% saponin in PBS according to the method of Willingham and Yamada (1979). They were then stained by the indirect method, using initially a 1:10 dilution of guinea pig antiprekeratin antiserum in PBS-saponin, then a 1:10 dilution of PBS-saponin of a rabbit anti-guinea pig IgG fraction conjugated with horseradish peroxidase (Nordic Immunological Laboratories, Tilburg). Cells were fixed again with 3% glutaraldehyde in PBS and rinsed in PBS for 30 min. Horseradish peroxidase was detected by diaminobenzidine(Graham and Karnovsky, 1966). After postfixation in 1% OsO4 in PBS for 30 min at 4°C, the cells were dehydrated and
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ET AL.
FIGS. l-6. Immunofluorescent staining of prekeratin in B. rossius spermatozoa. In Figs. 2 and 4 the complete tail region appears uniformly marked by the antiprekeratin antibody, while the head (arrows) is unstained (compare with the phase-contrast micrographs in Figs. 1 and 3). In Fig. 5 a control spermatozoon treated with a normal guinea pig serum is shown at phase contrast; no fluorescence is visible (Fig. 6). x 400.
embedded as described above. Alternatively, spermatozoa processed as described for immunofluorescence microscopy, were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 12 h at 4°C and processed for electron microscopy, according to Sharp et al. (1982).
RESULTS
Indirect immunofluorescence microscopy using antibodies directed against mammalian prekeratin, and particularly affinity purified antibodies, revealed bright staining
89
KERATIN-LIKE FILAMENTS IN INSECT SPERMATOZOA
of the whole-tail region of the spermatozoon (Figs. 1-4). This staining was assumed to be specific, since it was not observed in control preparations incubated with preimmane guinea pig serum instead of prekeratin antibody containing serum (Figs. 5, 6). Using antibodies against other components of intermediate-sized filaments, vimentin and desmin, no distinct staining could be localized (not shown). In agreement with the morphological staining pattern, we found after immunoblotting a cross-reaction of the same prekeratin antibodies with two polypepetides present in a total lysate of sperm cells of B. rossius (Fig. 7). They are minor components, since they do not stain with Coomassie blue. The molecular weights of these prekeratin-like polypeptides are a b o u t 60 000 and 68 000 Da and thus within the range o f known vertebrate prekeratins (Franke et aL, 1981; Moll et aL, 1982). Electron microscopic examination revealed two longitudinal elongate organelles in the whole length of the tail region (Fig. 8). They are made up by four juxtaposed laminae, longitudinally oriented with respect to the flagellar major axis. These four laminae are separated from one another by a space 10 nm wide, and consist of longitudinally oriented filaments lying at 10-rim intervals to one another. The 10-nm filaments are particularly evident after sonication and negative staining (Fig. 10). This array of filaments remains well preserved even after treatment with nonionic detergent Triton X-100 and high ionic strength solutions (Fig. 9). When saponin-treated spermatozoa were incubated with antiprekeratin antibodies and subsequently revealed by indirect staining with horseradish peroxidase, an intense staining over the crystalline bodies became visible (Fig. 11). Control incubations remained unstained (Fig. 12). Spermatozoa treated with prekeratin and FITC-conjugated antibodies revealed the same staining pattern at the level of the two crystalline
a
b
c
d
e
m
FIG. 7. Immunologic reaction of total extract of Bacillus sperm resolved by polyacrylamide gel electrophoresis (bands b and d), blotted on nitrocellulose sheets and incubated either with prekeratin antibodies against bovine prekeratin (c) or with normal rabbit serum as a control (e). Note (*) specific reaction of two minor bands from total sperm extract with prekeratin antibodies (c). (a) Markers: V, vimentin; A, Actin.
organelles (Fig. 13) not visible in the controls (Fig. 14). We therefore conclude that the proteins which are detected by crossreacting prekeratin antibodies, are localized in the crystalline bodies of the insect sperm tail. DISCUSSION
We have presented evidence that sperm cells of the insect B. rossius contain two polypeptides which specifically react with keratin antibodies, thus appear to share common antigenic determinants with vertebrate cytokeratins. Moreover, they have the molecular weight within the range of mammalian prekeratins (Franke et al., 1981; Moll et al,, 1982), and form 10-nm filaments, which are insoluble in nonionic detergent and at high ionic strength. In order to localize these polypeptides in the sper-
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W
r p
FIG. 8. Spermatozoon of B. rossius. Cross section of the tail region showing the two enormous crystalline accessory bodies (b) flanking the axoneme (a). FIG. 9. The same region in a Triton-extracted sperm. Accessorybodies (b) appear unchanged, x 100 000. Fit. 10. Negativestaining with PTA of an accessory body ofB. rossius sperm tail. The body appears as a bundle of 10-nm filaments. × 75 000.
m a t o z o o n , we used i m m u n o h i s t o c h e m i c a l methods, at both the light and electron microscopic level. With these methods, we localized these prekeratin-like polypeptides in the crystalline accessory bodies o f the sperm tail, which in fact consist o f a packing o f 10-nm filaments. These bodies are a characteristic o f the s p e r m a t o z o o n o f several insects, they flank the a x o n e m e t h r o u g h o u t almost its whole length, and are particularly
conspicuous in Bacillus, giving to the flagellar m o v e m e n t a pattern o f a double series o f waves (Baccetti et al., 1973). A l t h o u g h i n t e r m e d i a t e - s i z e d filaments have not been as systematically studied in in invertebrates as has been done in higher vertebrates (Osborn and Weber, 1982), they appear to be morphologically similar to, and might share biochemical characteristics with vertebrate 10-nm filaments. For instance,
KERATIN-LIKE FILAMENTS IN INSECT SPERMATOZOA
91
@
@ FIG. 11. Spermatozoon of B. rossius. Cross section of the tail showing accessory bodies (b) strongly stained by the DAB--peroxidase method after antiprekeratin serum incubation, x 100 000. FIG. 12. Control to the above method, after normal guinea pig serum incubation, x 100 000. FIG. 13. Same material, with accessory bodies (b) strongly stained by the FITC-conjugated anti-guinea pig IgG after anti-prekeratin serum incubation, x 100 000. FIG. 14. Control to the above method, after normal guinea pig serum incubation, x 100 000. K c cells of Drosophila melanogaster c o n t a i n h e a t - s e n s i t i v e p r o t e i n s w h i c h a p p e a r to b e constituents of a filamentous cytoplasmic m e s h w o r k ( F a l k n e r et al., 1981). M o n o c l o n a l a n t i b o d i e s r a i s e d a g a i n s t these Dro-
sophila p r o t e i n s c r o s s - r e a c t w i t h v i m e n t i n a n d d e s m i n o f m a m m a l i a n c u l t u r e d cells i n d i c a t i n g the p r e s e n c e o f at least s o m e c o m m o n d e t e r m i n a n t s i n these m o l e c u l e s ( F a l k n e r et aL, 1981). T h e p r e s e n c e o f p r e -
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keratin-like polypeptides in spermatozoa o f insects m a y demonstrate that also this group o f proteins, yet displaying p r o n o u n c e d heterogeneity a m o n g m a m m a l s (Franke et al., 1981; Moll et al., 1982), contains some domains firmly conserved during evolution and which are c o m m o n to similar polypeptides in animals as far apart as insects and m a m m a l s . Cytokeratins seem not to be confined in insect sperm: in the same class, tracheal taenidia contain filaments which also can be decorated with keratin antibodies (Baccetti et al., 1984). The failure o f detecting sequences h o m o l o g u e s to c - D N A specific for m a m m a l i a n keratins in Drosophila reported by Fuchs et al. (1981) seemed to be in apparent contrast with our data. But this is due to the fact that the D N A sequence coding for keratins or keratin-like molecules have diverged sufficiently during evolution, so that under conditions o f high hybridization stringency, a m a m m a l i a n probe does not recognize insect sequences. In fact m o r e recently the same authors (Fuchs and Marchuk, 1983) have been able to extend to the g e n o m e o f Drosophila (and o f yeast) sequences related to h u m a n kerRtins cDNAs. It means that the proteins themselves can have remained sufficiently conserved during animal evolution to allow recognition o f at least some antigenic determinants by an antibody raised against bovine keratin.
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